Biological membranes, proteins, subcellular organelles, and other samples are being investigated via atomic force microscopy (AFM), Raman and fluorescent spectroscopy, and related biophysical approaches in this project covering several research collaborations. With further expansion of our technology, instrumentation, and data analysis methods, we have advanced a number of these applications in collaborations with many NIH intramural and extramural researchers. Major collaborations include:[unreadable] [unreadable] (1) We conpleted work on the effect of a mycobacterium tuberculosis-derived lipid, Lipoarabinomannan[unreadable] (LAM), on biological membranes with Drs. E. Hayakawa, F. Tokumasu, J.A. Dvorak (NIAID/NIH), and coworkers. Lipoarabinomannan is an integral component of the cell wall of the acid-fast bacillus mycobacterium tuberculosis (MTB). One hallmark of a tuberculosis infection is the ability of the bacterium to subvert the normal macrophage defense mechanism of the host immune response. LAM is reported to be involved in the inhibition of a phago-lysosomal fusion step of this defense mechanism. We have produced membranes in vitro composed of LAM and varying lipids to simulate phagosomal lipid membranes and quantified the effects of LAM using AFM and related studies. We found that LAM markedly alters membrane nanostructure and inhibits large unilamellar vesicle fusion, with possible implications for the survival of MTB and the fighting of uman tuberculosis disease.[unreadable] [unreadable] (2) We have investigated the macromolecular structure of a recombinant Plasmodium falciparum Merozoite Surface Protein 3 (MSP3) and more recently Plasmodium falciparum circumsporozoite protein (CSP) via AFM in collaboration with Dr. David Narum (NIAID, NIH) and coworkers. MSP3 and CSP are potential components of a human malaria vaccine. These protein antigens are being produced from Escherichia coli, purified, and characterized in a manner suitable for scale-up toward human trials. We have focused on using AFM and related studies to understand the structural properties of these antigens under a range of solution conditions, finding mainly EcCSP monomers, but EcMSP3 monomers, dimers, and multimers with fluctuating folding of the secondary structural domains. Such structures should have implications for protein-protein interactions and human immunological response. [unreadable] [unreadable] (3) We have expanded our AFM studies of clathrin and clathrin coated vesicles (CCVs) in collaboration with Drs. Ralph Nossal (NICHD, NIH), Eileen Lafer (Univ. Texas Health Sciences Center, San Antonio) and coworkers. Clathrin triskelia form the outer clathrin lattice cages of the CCVs during subcellular trafficking via interactions with adaptor proteins, membrane lipids, and other cofactors. The intricacies of these dynamic macromolecular constructs have inspired numerous structural and functional studies. Here we have continued to develop and apply new schemes of atomic force microscopy (AFM) and related analyses toward the characterizations of triskelia and their assemblies. We have resolved variable profiles of triskelia on mica surfaces for the first time by AFM at a resolution comparable to electron microscopy. Classical tri-leg, filamentous pin-wheel shapes, as well as non-planar triskelion conformations and dimers, are readily observed both dried on mica surface and showing shape flexibility under buffers. Pentagonal and hexagonal lattice structures are well visualized in a variety of clathrin assemblies with or without AP180 adaptors, similar to those of the native CCVs purified from bovine brains. We have detected a considerable variability in triskelion conformation and clathrin cage nano-mechanics. We have also produced single molecule force spectroscopy (SMFS) of triskelia and CCVs under buffers and revealed, also for the first time, a series of internal energetic barriers that[unreadable] characterize triskelion heavy chain folding and unfolding, including molecular sequence and structure periodicity for both the seven repeating 145aa motifs and numerous 30aa hairpins. The dynamic stability of these domains has been obtained. [unreadable] [unreadable] (4) We have continued our collaboration with Drs. Shui-Lin Niu, Drake Mitchell, Klaus Gawrisch (NIAAA, NIH) and colleagues, using AFM to characterize at sub-nanometer resolution the structure and function of the protein rhodopsin, a G-protein coupled receptor (GPCR) of the visual pathway, in native rod outer segment membranes and in reconstituted lipid membranes. We are introducing new AFM modalities, including a new Raman-AFM spectroscopy instrument, to further reveal rhodopsin molecule organization and to explore the connection between rhodopsin signaling and the lipid membrane environment. We are optimizing the Raman-AFM detecting sensitivity toward single bilayer nanometric domain characterization of proteins and lipids using resonance and metal surface enhancements, novel AFM tips, and measurement modality. We aim to achieve better correlations between GPCR function and membrane structure at nanometric scale.[unreadable] [unreadable] We are also conducting other AFM-related investigations in certain areas relating to live cells, bacterial biofilms, biomimicking materials, and fundamental surface sciences.